Orphan Granzymes Downstream from Granzyme B Are Important for Tumor Clearance In Vivo and in Vitro.

Abstract Cytotoxic lymphocytes (Natural Killer cells and Cytotoxic T lymphocytes) can utilize the perforin/granzyme pathway as a major mechanism to kill pathogen-infected cells and tumor cells. Perforin is responsible for delivering and/or trafficking the granzymes (a family of neutral serine proteases) to the target cells. In the target cell cytoplasm and nucleus, the granzymes deliver the lethal hits. Granzymes A and B are the best characterized granzymes, and they can cleave a variety of important protein substrates to execute the target cells. However, some tumors and viruses have developed potent granzyme inhibitors that may allow them to evade cytotoxic lymphocyte-induced death. Interestingly, additional granzyme genes downstream from granzyme B (C, F, G, and D) on murine chromosome 14 are also expressed in cytotoxic lymphocytes, and are referred to as “orphans” since their functions have not been defined. We have developed two kinds of granzyme B knockout mice in the 129/SvJ background (H-2b) and examined their expression of granzyme B and orphan granzymes using quantitative RT-PCR and Western Blotting. In the first mouse (Gzm B−/−/+PGK-neo) a PGK-neo cassette was retained in the granzyme B gene, which caused a neighborhood effect, with significantly reduced expression of orphan granzymes C and F in cytotoxic lymphocytes (this mouse is referred to as “B cluster” deficient); In the second mouse (Gzm B−/−/ΔPGK-neo) the PGK-neo cassette was removed by Cre/loxP technology, which restored expression of granzymes C and F in cytotoxic lymphocytes (referred to as “B only” deficient). Both mutations completely abolish granzyme B expression. Using a Flow-based Killing Assay (FloKA), we have examined the cytotoxic functions of lymphocytes derived from mixed lymphocyte reactions (MLR) and 10-day lymphokine activated killer (LAK) cultures. We have found that granzyme B cluster-deficient cytotoxic lymphocytes (H-2b) generated by MLR kill allogeneic P815 or TA-3 tumor cells (H-2d) less efficiently than those deficient for granzyme B only (e.g. P815 killing at 3 hours, WT: 35%±1%, B only-deficient: 24%±5%, B cluster-deficient: 14%±3%, p<0.001). The reduction in granzyme B cluster-deficient killing is also seen with LAK cells against YAC-1 and RMA-S target cells (e.g. RMA-S killing at 4 hours, WT: 26%±1%, B only-deficient: 24%±1%, B cluster-deficient: 18%±1%, p<0.001). These results suggest that both allogeneic CTL and LAK cells require orphan granzymes (C and/or F) for optimal tumor cell killing. The defects in cytotoxicity detected by the FloKA assay have been confirmed to be biologically relevant (Revell et al, Blood2003, 102 (11): 1022) since granzyme B cluster-deficient mice cleared P815 cells less efficiently than either WT or granzyme B only-deficient mice (p<0.02). These studies suggest that the orphan granzymes are important for cytotoxic lymphocyte functions, and that they may provide a source of functional redundancy that would help protect from pathogens or tumor cells that express inhibitors of granzyme A or B.